Methods for screening variant of target gene

ABSTRACT

Provided are methods for screening a desired variant of a target gene in a eukaryotic system. Compositions for screening a desired variant of a target gene are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 16/968,602,filed Aug. 9, 2020, which is the national phase of InternationalApplication PCT/US2019/017375, filed Feb. 8, 2019, which claims priorityto U.S. provisional patent application No. 62/628,236, filed Feb. 8,2018, the disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention generally relates to compositions and methods forscreening a desired variant of a target gene.

SEQUENCE LISTING

An official copy of the sequence listing is submitted concurrently withthe specification electronically via EFS-Web as an ASCII formattedsequence listing with a file name of 044903-8023WO01_ST25, a creationdate of Feb. 8, 2019, and a size of about 4.08 kilobytes. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

BACKGROUND

Variant expression library platforms provide powerful tools to study therelationship between genotype and phenotype. In a typical variantexpression library-based assay, tens or hundreds of thousands ofvariants are introduced into a model system, a selection pressure isapplied, and the changes in the frequency of the variants duringselection are measured. This approach has been used to measure theeffect of all possible gene deletions in a genome (Shalem O et al.,Genome-scale CRISPR-Cas9 knockout, Science (2014) 343:84-88; Want T etal., Genetic screens in human cells using the CRISPR-Cas9 system,Science (2014) 343:80-84) or all possible single mutants of a protein(Fowler D M and Fields S, Deep mutational scanning a new style ofprotein science, Nat Methods (2014) 11:801-807). The variant expressionlibrary platforms can also be used to screen variant with desiredphenotype, e.g., an enzyme variant with altered specificity or enhancedactivity, which has enormous applications in bioengineering.

Variant expression library-based assays require that each cell ororganism contain a defined genetic alteration, which must remain stablethroughout the experiment. While certain system, such as bacteria andyeast, can meet these requirement by transforming each cell with asingle plasmid, it has been difficult to establish a mammalian system(e.g., using human cells) that can introduce single and stable variantper cell at the required scale. Plasmid transfection, the simplestoption, results in the unstable introduction of hundreds or thousands ofplasmids into each cell. Virus (e.g., Lentivirus) infection at lowtiters, though resulting in stable integration of a single variant insome cells, exhibits widely varying expression due to random viralintegration that increases noise and confounds comparisons. CRISPR/Cassystem provides an option that avoids the problems of multiple variantsper cell and random insertion, but are limited by the low knock-inefficiency, especially for variants of large size. Therefore, there is aneed for new systems and methods for mammalian cell based variantexpression library platforms.

SUMMARY

The present disclosure in one aspect provides a method for screening adesired variant of a target gene. In one embodiment, the methodcomprises (1) obtaining a cell line comprising at a genomic locus afirst unidirectional recombination site recognized by a site-specificunidirectional recombinase; (2) generating a cell library by (i)introducing to the cell line a library of nucleic acid constructs, eachof the nucleic acid constructs comprising: (a) a second unidirectionalrecombination site recognized by the site-specific unidirectionalrecombinase, and (b) a variant of a target gene, wherein at least one ofthe nucleic acid constructs comprises a desired variant of the targetgene, (ii) expressing in the cell line the site-specific unidirectionalrecombinase, and (iii) maintaining the cell line under conditions thatfacilitate recombination between the first and the second unidirectionalrecombination sites mediated by the site-specific unidirectionalrecombinase; and (3) selecting from the cell library a cell comprisingin its genome the desired variant of the target gene.

In certain embodiments, the specific genomic locus is Hipp11 (H11)locus.

In certain embodiments, the site-specific unidirectional recombinase isBxb1 integrase. In certain embodiments, the Bxb1 integrase is expressedusing a construct comprising polynucleotide sequence of SEQ ID NO: 1.

In certain embodiments, the cell line further comprises at the genomiclocus: a first promoter and a second promoter. In certain embodiments,the first promoter is a Tet-on promoter. In certain embodiments, thesecond promoter is an EF-1 alpha promoter.

In certain embodiments, the cell line is derived from a HEK293 cell.

In certain embodiments, the target gene is an enzyme. In certainembodiments, the target gene is a second site-specific unidirectionalrecombinase. In certain embodiments, the second site-specificunidirectional recombinase is phiC31 integrase. In certain embodiments,the variant of the second site-specific unidirectional recombinaserecognizes a variant unidirectional recombination site.

In certain embodiments, the selecting step comprises

-   -   introducing a selection construct to the cell library, wherein        the selection construct comprises        -   a third unidirectional recombination site,        -   a third promoter,        -   a fourth unidirectional recombination site, and        -   a selectable marker,        -   wherein at least one of the third and the fourth            unidirectional recombination sites is a variant that is not            recognized by the second site-specific unidirectional            recombinase but recognized by the desired variant of the            second site-specific unidirectional recombinase, and        -   wherein the third promoter and the selectable marker is            arranged in opposite orientation,    -   maintaining the cell library under conditions that facilitate        recombination between the third and the fourth unidirectional        recombination sites mediated by the desired variant of the        second site-specific unidirectional recombinase, thereby        reversing the orientation of the third promoter or the        selectable marker in the selection construct, and    -   selecting the cell expressing the selectable marker.

In certain embodiments, the target gene is a Cas protein.

In certain embodiments, the variant of the Cas protein recognizes avariant protospacer adjacent motif (PAM), or has higher on-targetspecificity or has lower immunogenicity.

In certain embodiments, the target gene is a virus capsid gene. Incertain embodiments, the virus capsid gene is an AAV capsid gene. Incertain embodiments, the variant of the virus capsid gene has betterpackaging ability of packaging>4.7kb DNA in size or increasedinfectivity to a target cell/tissue.

In certain embodiments, the method further comprises generating from thecell a nucleic acid that encodes the desired variant of the target gene.

In another aspect, the present disclosure provides a cell selectedaccording to the method described herein.

In another aspect, the present disclosure provides a nucleic acidgenerated according to the method described herein.

In yet another aspect, the present disclosure provides a kit forgenerating a desired variant of a target gene. In one embodiment, thekit comprises: (i) a cell line comprising at a genomic locus: a firstunidirectional recombination site recognized by a site-specificunidirectional recombinase; (ii) a nucleic acid construct comprising asecond unidirectional recombination site recognized by the site-specificunidirectional recombinase; and (iii) a reagent for generating a variantlibrary of a target gene.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings, which are incorporated herein, form part ofthe specification. Together with this written description, the drawingsfurther serve to explain the principles of, and to enable a personskilled in the relevant art(s), to make and use the present invention.

FIG. 1 illustrates the methods for evaluating the high efficiencyintegration efficiency of loci provided in the present disclosure.

FIG. 2 illustrates the nucleotide sequence of codon-optimized Bxb1 genewith a bi-partite Xenopus NLS.

FIG. 3 illustrates the high integration efficiency of usingcodon-optimized Bxb1 gene with a bi-partite Xenopus NLS.

FIG. 4 illustrates the sequences of target Site A and a series ofintermediate Site A.

FIG. 5 illustrates the construct containing variant phiC31 integrasegene.

FIG. 6 illustrates the generation of cell library containing variants ofan integrase.

FIG. 7 illustrates a method of screening from the cell library of FIG. 6an integrase variant that recognizes a variant pseudo-recombinationsite.

FIG. 8 illustrates a method of screening a mutant AAV capsid gene thatcan better package a larger sized DNA.

DESCRIPTION OF THE INVENTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

DEFINITION

The following definitions are provided to assist the reader. Unlessotherwise defined, all terms of art, notations and other scientific ormedical terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the chemical andmedical arts. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over the definition of the term asgenerally understood in the art.

As used herein, the singular forms “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise.

A “cell”, as used herein, can be prokaryotic or eukaryotic. Aprokaryotic cell includes, for example, bacteria. A eukaryotic cellincludes, for example, a fungus, a plant cell, and an animal cell. Thetypes of an animal cell (e.g., a mammalian cell or a human cell)includes, for example, a cell from circulatory/immune system or organ,e.g., a B cell, a T cell (cytotoxic T cell, natural killer T cell,regulatory T cell, T helper cell), a natural killer cell, a granulocyte(e.g., basophil granulocyte, an eosinophil granulocyte, a neutrophilgranulocyte and a hypersegmented neutrophil), a monocyte or macrophage,a red blood cell (e.g., reticulocyte), a mast cell, a thrombocyte ormegakaryocyte, and a dendritic cell; a cell from an endocrine system ororgan, e.g., a thyroid cell (e.g., thyroid epithelial cell,parafollicular cell), a parathyroid cell (e.g., parathyroid chief cell,oxyphil cell), an adrenal cell (e.g., chromaffin cell), and a pinealcell (e.g., pinealocyte); a cell from a nervous system or organ, e.g., aglioblast (e.g., astrocyte and oligodendrocyte), a microglia, amagnocellular neurosecretory cell, a stellate cell, a boettcher cell,and a pituitary cell (e.g., gonadotrope, corticotrope, thyrotrope,somatotrope, and lactotroph); a cell from a respiratory system or organ,e.g., a pneumocyte (a type I pneumocyte and a type II pneumocyte), aclara cell, a goblet cell, an alveolar macrophage; a cell from circularsystem or organ, e.g., myocardiocyte and pericyte; a cell from digestivesystem or organ, e.g., a gastric chief cell, a parietal cell, a gobletcell, a paneth cell, a G cell, a D cell, an ECL cell, an I cell, a Kcell, an S cell, an enteroendocrine cell, an enterochromaffin cell, anAPUD cell, a liver cell (e.g., a hepatocyte and Kupffer cell); a cellfrom integumentary system or organ, e.g., a bone cell (e.g., anosteoblast, an osteocyte, and an osteoclast), a teeth cell (e.g., acementoblast, and an ameloblast), a cartilage cell (e.g., a chondroblastand a chondrocyte), a skin/hair cell (e.g., a trichocyte, akeratinocyte, and a melanocyte (Nevus cell), a muscle cell (e.g.,myocyte), an adipocyte, a fibroblast, and a tendon cell), a cell fromurinary system or organ (e.g., a podocyte, a juxtaglomerular cell, anintraglomerular mesangial cell, an extraglomerular mesangial cell, akidney proximal tubule brush border cell, and a macula densa cell), anda cell from reproductive system or organ (e.g., a spermatozoon, aSertoli cell, a leydig cell, an ovum, an oocyte). A cell can be normal,healthy cell; or a diseased or unhealthy cell (e.g., a cancer cell). Acell further includes a mammalian zygote or a stem cell which include anembryonic stem cell, a fetal stem cell, an induced pluripotent stemcell, and an adult stem cell. A stem cell is a cell that is capable ofundergoing cycles of cell division while maintaining an undifferentiatedstate and differentiating into specialized cell types. A stem cell canbe an omnipotent stem cell, a pluripotent stem cell, a multipotent stemcell, an oligopotent stem cell and a unipotent stem cell, any of whichmay be induced from a somatic cell. A stem cell may also include acancer stem cell. A mammalian cell can be a rodent cell, e.g., a mouse,rat, hamster cell. A mammalian cell can be a lagomorpha cell, e.g., arabbit cell. A mammalian cell can also be a primate cell, e.g., a humancell.

A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide,for example, in vivo when placed under the control of appropriateregulatory sequences (or “control elements”). The boundaries of thecoding sequence are typically determined by a start codon at the 5′(amino) terminus and a translation stop codon at the 3′ (carboxy)terminus. A coding sequence can include, but is not limited to, cDNAfrom viral, procaryotic or eucaryotic mRNA, genomic DNA sequences fromviral or procaryotic DNA, and even synthetic DNA sequences. Atranscription termination sequence may be located 3′ to the codingsequence. Other “control elements” may also be associated with a codingsequence. A DNA sequence encoding a polypeptide can be optimized forexpression in a selected cell by using the codons preferred by theselected cell to represent the DNA copy of the desired polypeptidecoding sequence.

“Derived from” in the context of an amino acid sequence orpolynucleotide sequence (e.g., a nucleic acid sequence “derived from” awild-type attP) is meant to indicate that the polypeptide or nucleicacid has a sequence that is based on that of a reference polypeptide ornucleic acid (e.g., a naturally occurring phiC31 integrase or encodingnucleic acid), and is not meant to be limiting as to the source ormethod in which the protein or nucleic acid is made. A first polypeptideis “derived from” a second polypeptide if it is (i) encoded by a firstpolynucleotide derived from a second polynucleotide encoding the secondpolypeptide, or (ii) displays sequence identity to the secondpolypeptides.

The “Hipp11 (H11) locus,” as used herein, refers to a “safe harbor”genomic locus that allows gene expression without disrupting internalgene function. In mic, the H11 locus is located within an intergenicregion between Eif4enif1 and Drg1 genes, which are mapped close to thecentromere of chromosome 11 (B Tasci et al. Proc Natl Acad Sci USA(2011) 108:7902-07). Human H11 locus is located on human chromosome22q12.2, between the DRG1 and EIF4ENIF1 genes (F Zhu et al. NucleicAcids Res (2014) 42:e34).

The term “introduce” in the context of inserting a nucleic acid sequenceinto a cell, means “transfection”, or ‘transformation”, or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell wherein the nucleicacid sequence may be present in the cell transiently or may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid, or mitochondrial DNA), converted into an autonomous replicon.The nucleic acid construct of the present disclosure may be introducedinto a cell using any method known in the art. Various techniques fortransforming animal cells may be employed, including, for example:microinjection, retrovirus mediated gene transfer, electroporation,transfection, or the like (see, e.g., Keown et al., Methods inEnzymology 1990, 185:527-537).

As used herein, “locus” refers to a specific location on a chromosome. Aknown locus can contain known genetic information, such as one or morepolymorphic marker sites.

The term “operably linked” refers to an arrangement of elements whereinthe components so described are configured so as to perform their usualfunction. In the case of a promoter, a promoter that is operably linkedto a coding sequence will direct the expression of the coding sequence.The promoter or other control elements need not be contiguous with thecoding sequence, so long as they function to direct the expressionthereof. For example, intervening untranslated yet transcribed sequencescan be present between the promoter sequence and the coding sequence andthe promoter sequence can still be considered “operably linked” to thecoding sequence.

The term “nucleic acid construct” means a nucleic acid sequence that hasbeen constructed to comprise one or more functional units not foundtogether in nature. Examples include circular, double-stranded,extrachromosomal DNA molecules (plasmids), cosmids (plasmids containingCOS sequences from lambda phage), viral genomes comprising non-nativenucleic acid sequences, and the like.

The term “overexpression” used herein refers to excessively high levelof gene expression that produces a gene-related phenotype. Theoverexpression of a gene can be resulted from increased copy number ofthe encoding nucleic acids, or increased level of mRNA transcripts orprotein, or decreased degradation of transcripts and/or protein.

As used herein, a “promoter” and “promoter-enhancer” sequence is anarray of nucleic acid control sequences to which RNA polymerase bindsand initiates transcription. A promoter includes necessary nucleic acidsequences near the start site of transcription, such as, in the case ofa polymerase II type promoter, a TATA element. A promoter-enhancer alsooptionally includes distal enhancer or repressor elements which can belocated as much as several thousand base pairs from the start site oftranscription. The promoter determines the polarity of the transcript byspecifying which DNA strand will be transcribed. Eukaryotic promotersare complex arrangements of sequences that are utilized by RNApolymerase II. General transcription factors (GTFS) first bind specificsequences near the start and then recruit the binding of RNA polymeraseII. In addition to these minimal promoter elements, small sequenceelements are recognized specifically by modularDNA-binding/trans-activating proteins (e.g., AP-1, SP-1) that regulatethe activity of a given promoter. Viral promoters serve the samefunction as bacterial or eukaryotic promoters and either provide aspecific RNA polymerase in trans (bacteriophage T7) or recruit cellularfactors and RNA polymerase (SV40, RSV, CMV). Promoters may be,furthermore, either constitutive or regulatable. Inducible elements areDNA sequence elements which act in conjunction with promoters and maybind either repressors or inducers. In such cases, transcription isvirtually “shut off” until the promoter is derepressed or induced, atwhich point transcription is “turned-on.” Examples of eukaryoticpromoters include, but are not limited to, the following: the promoterof the mouse metallothionein I gene sequence (Hamer et al., J. Mol.Appl. Gen. (1982) 1:273-288); the TK promoter of Herpes virus (McKnight,Cell (1982) 31:355-365); the SV40 early promoter (Benoist et al., Nature(1981) 290:304-310); the yeast gall gene sequence promoter (Johnston etal., Proc. Natl. Acad. Sci. (1982) 79:6971-6975); Silver et al., Proc.Natl. Acad. Sci. (1984) 81:5951-59SS), the CMV promoter, the EF-1promoter, Ecdysone-responsive promoter(s), tetracycline responsivepromoter, and the like.

In general, a “protein” is a polypeptide (i.e., a string of at least twoamino acids linked to one another by peptide bonds). Proteins mayinclude moieties other than amino acids (e.g., may be glycoproteins)and/or may be otherwise processed or modified. Those of ordinary skillin the art will appreciate that a “protein” can be a completepolypeptide chain as produced by a cell (with or without a signalsequence), or can be a functional portion thereof. Those of ordinaryskill will further appreciate that a protein can sometimes include morethan one polypeptide chain, for example linked by one or more disulfidebonds or associated by other means.

A “pseudo-recombination site” is a DNA sequence recognized by arecombinase enzyme such that the recognition site differs in one or morebase pairs from the wild-type recombinase recognition sequence and/or ispresent as an endogenous sequence in a genome that differs from thegenome where the wild-type recognition sequence for the recombinaseresides.

“Pseudo attP site” or “pseudo attB site” refer to pseudo sites that aresimilar to wild-type phage or bacterial attachment site sequences,respectively, for phage integrase enzymes. “Pseudo att site” is a moregeneral term that can refer to either a pseudo attP site or a pseudoattB site.

As used herein, the term “recombinase” or “site-specific recombinase”refers to a family of highly specialized enzymes that promote DNArearrangement between specific target sites (Greindley et al., 2006;Esposito, D., and Scocca, J. J., Nucleic Acids Research 25, 3605-3614(1997); Nunes-Duby, S. E., et al, Nucleic Acids Research 26, 391-406(1998); Stark, W. M., et al, Trends in Genetics 8, 432-439 (1992)).Virtually all site-specific recombinases can be categorized within oneof two structurally and mechanistically distinct groups: the tyrosine(e.g., Cre, Flp, and the lambda integrase) or serine (e.g., phiC31integrase, gamma-delta resolvase, Tn3 resolvase and Gin invertase)recombinases. Both recombinase families recognize target sites composedof two inversely repeated binding elements that flank a spacer sequencewhere DNA breakage and religation occur. The recombination processrequires concomitant binding of two recombinase monomers to each targetsite: two DNA-bound dimers (a tetramer) then join to form a synapticcomplex, leading to crossover and strand exchange. In particular,recombinases can recognize endogenous sequences in a genome of interest.

“Uni-directional recombinases” or “integrases” refer to recombinaseenzymes whose recognition sites are destroyed after the recombinationhas taken place. In other words, the sequence recognized by therecombinase is changed into one that is not recognized by therecombinase upon recombination. As a result, once a sequence issubjected to recombination by the uni-directional recombinase, thecontinued presence of the recombinase cannot reverse the previousrecombination event.

As used herein, a “selection marker” or “selectable marker” refers agene whose expression in cells allows the cells to be enriched ordepleted under particular culture conditions. A selectable marker may bea foreign gene or a cellular gene which is not naturally expressed orsuch a gene which is naturally expressed, but at an inappropriate level,in the target cell populations. If the expression of the gene allows thecells to be enriched under particular conditions, the selectable markeris a “positive selectable marker.” Typically, a positive selectablemarker is a gene that encodes for antibiotic resistance and selectingfor those cells that express the selection marker comprises introducingantibiotic into the culture. In use, application of the antibioticselectively kills or ablates cells that do not express the marker,leaving behind a population of cells purified or enriched in respect ofthose expressing the antibiotic resistance. Examples of a positiveselectable marker include aminoglycoside phosphotransferase (neomycinresistance gene), puromycin-N-acetyl transferase (puromycin resistancegene), hygromycin resistance gene, and blasticidin S deaminase(blasticidin S resistance gene). Other examples of positive selectablemarker include genes that can be used to select through cell sorting,e.g., fluorescent proteins, e.g., GFP and RFP, and cell surface markers.Conversely, if the expression of the gene allows the cells to bedepleted under particular culture condition, the selectable marker is a“negative selectable marker.” Examples of a negative selectable markerinclude thymidine kinase gene. In use, application of ganciclovir killsthe cells with expression of thymidine kinase. Other examples ofnegative selectable markers include DT toxin, cell death genes, such asTRAIL, caspases and BCL2 family genes.

The term “sequentially” when used to describe two polynucleotidesequences means that the two sequences are not overlap, while the firstsequence can either locate at the upstream (5′) or downstream (3′) ofthe second sequence.

The terms “subject” and “patient” as used herein are interchangeable. Asubject is an animal, preferably a mammal such as a non-primate (e.g.,cows, pigs, horses, cats, dogs, rats, mice, etc.) and a primate (e.g.,monkey (e.g., a rhesus monkey, a cynomolgus monkey or chimpanzee) andhuman), and most preferably a human.

“Transcript” refers to a mRNA formed by the gene transcription forprotein expression. One or more transcripts variants are formed from thesame DNA segment via differential splicing. In such a process,particular exons of a gene may be included within or excluded from themessenger mRNA (mRNA), resulting in translated proteins containingdifferent amino acids and/or possessing different biological functions.

The term “transgene” refers to an exogenous polynucleotide introducedinto a host cell (e.g. a HEK293 cell), irrespective of the method usedfor the introduction. The methods include those known in the art,including vector-mediated gene transfer (by, e.g., viralinfection/transfection, or various other protein-based or lipid-basedgene delivery complexes) as well as techniques facilitating the deliveryof “naked” polynucleotides (such as electroporation, “gene gun” deliveryand various other techniques used for the introduction ofpolynucleotides).

The term “variant,” when used in conjunction with a gene, a nucleotidesequence or a protein, refers to a gene, a nucleotide sequence orprotein that is different from the reference or original gene,nucleotide sequence or protein in at least one nucleotide or amino acidresidue. In certain circumstances, the term “variant” is usedinterchangeably with the term “mutant.”

Method of Screening Variant of Target Gene

The present disclosure in one aspect relates to methods of screeningdesired variants of a target gene.

As used herein, “screen” refers to an experimental technique used toidentify and select for variants of a gene that possess a phenotype orfunction of interest in a variants population.

As used herein, a “gene” refers to a sequence of DNA or RNA that codesfor a molecule, e.g., a polypeptide or RNA, having a function. Duringgene expression, the DNA is first transcribed into RNA, which can bedirectly functional or act as an intermediate template for a polypeptidethat performs a function. Genes can acquire mutations in their sequence,resulting in different variants. Each variant may encode a slightlydifferent version of a polypeptide, which performs different function.

In one embodiment, the screen method comprises obtaining a cell linewhich comprises at a genomic locus a first unidirectional recombinationsite recognized by a site-specific unidirectional recombinase. In someembodiments, the screen method further comprises generating a celllibrary using the cell line that comprises the unidirectionalrecombination site and a library of nucleic acid constructs, each of thenucleic acid constructs comprising a second unidirectional recombinationsite recognized by the site-specific unidirectional recombinase, and avariant of the target gene.

In some embodiments, the genomic locus containing the recombination siteis a region that provides increased expression of transgene contained inthe region. Examples of such locus include without limitation, ROSA26,ROSA26 like locus, HPRT, AAVS1 and Hipp11 (H11). In a preferredembodiment, the locus is H11.

Recombination Sites

Recombination sites used in the present methods include those recognizedby unidirectional, site-directed recombinases, such as Bxb1, phiC31,TP901-1, and R4, wild-type or variations thereof. Any sites recognizedby recombinases described in US 2010/0190178 may also be used.

Binding sites for phage integrase enzymes as recombinases, such as theBxb1 integrase, are traditionally called attB and attP (i.e., the targetsites of the integrase). These sites have a minimal length ofapproximately 34-40 base pairs (bp) (Groth A C et al., Proc. Natl. Acad.Sci. USA 97, 5995-6000 (2000)). These sites are typically arranged asfollows: AttB comprises a first DNA sequence attB5′, a core region, anda second DNA sequence attB3′ in the relative order attB5′-coreregion-attB3′. AttP contains a first DNA sequence (attP5′), a coreregion, and a second DNA sequence (attP3′) in the relative orderattP5′-core region-attP3′. The recombinase mediates production ofrecombination-product sites that can no longer act as substrates for therecombinase. The recombination-product sites contain, for example, therelative order attB5′-recombination-product site-attP3′ andattP5′-recombination-product site-attB3′. Where the recombination sitesare recognized by Bxb1, the sites can be variants of the nativeattP/attB sequences, such as tandem repeats (e.g., three repeats such asattPx3), truncated sequences, or both. In some embodiments, the firstrecombination site and the second recombination site are attP and attB,respectively, or vice versa.

Recombination sites used in the methods of the present disclosure mayalso include, e.g., bidirectional recombination sites, such FRTrecombination sites or loxP recombination sites.

Cell Line Containing Recombination Site

As used herein, a “cell line” refers to a cell culture derived from asingle cell and therefore consisting of a plurality of cells with auniform genetic makeup. In some embodiments, the cell line used in themethod is a mammalian cell line. In one embodiment, the cell line isderived from a HEK293 cell.

The method of generating a cell line containing the recombination siteof interest in a target locus is known in the art. See, e.g., Duportet Xet al., “A platform for rapid prototyping of synthetic gene networks inmammalian cells.” Nucleic Acids Res. (2014) 1; 42(21):13440-51; andMatreyek K et al., “A platform for functional assessment of largevariant libraries in mammalian cells” Nucleic Acids Res (2017)45(11):e102.

Typically, a nucleic acid construct comprising the recombination site ofinterest flanked by homology arms of the target locus is created. Thenucleic acid construct may also include additional nucleic acidfragments that facilitate the generation of the cell line, e.g.,selection marker sequences. In one embodiment, the nucleic acidconstruct contains a hygromycin resistance marker.

In some embodiments, the nucleic acid construct may also includeadditional nucleic acid fragments that facilitate selection of variantsof a target gene, such as promoter sequences, which will be inserted tothe target locus together with the recombination site. In someembodiments, the nucleic acid construct comprises a tetracycline (Tet)responsive promoter and a EF-1 promoter.

When the nucleic acid construct containing the recombination site ofinterest is introduced into a cell, the recombination site can beinserted into the target locus through homologous recombination. Incertain embodiments, a site-specific nuclease is expressed in the cellto generate a double strand break in order to increase the efficiency ofhomologous recombination. In some embodiments, the site-specificnuclease is a CRISPR/Cas protein, a zinc finger nuclease (ZFN) or atranscriptional activator-like effector nuclease (TALEN).

Variant Library

The cell line that comprises the first unidirectional recombination siteis used to generate a cell library using a library of nucleic acidconstructs. In some embodiments, each of the nucleic acid constructscomprises a second unidirectional recombination site recognized by thesite-specific unidirectional recombinase, and a variant of the targetgene.

Methods of generating variants of a target gene is known in the art.See, e.g., Zhou Y H et al., “Random mutagenesis of gene-sized DNAmolecules by use of PCR with Taq DNA polymerase.” Nucleic Acids Res.(1991)19(21):6052; Engler C et al., “Golden Gate Shuffling: A One-PotDNA Shuffling Method Based on Type IIs Restriction Enzymes” PLoS One.2009; 4(5):e5553; and Ashraf M et al., “ProxiMAX randomization: a newtechnology for non-degenerate saturation mutagenesis of contiguouscodons.” Biochem Soc Trans. (2013) 41(5):1189-94.

Zhou Y H et al has reported a simple method of random mutagenesis usingTaq DNA polymerase, which lacks a 3′-5′ exonucleolytic editing activityand thus becomes error-prone (Nucleic Acids Res. (1991)19(21):6052).Engler C et al developed a protocol to assemble multiple DNA fragmentstogether into a vector, allowing the generation of libraries ofrecombinant genes by combining several fragment sets prepared fromdifferent parental templates (PLoS One. 2009; 4(5):e5553). The protocolcan shuffle the DNA fragments derived from templates having no homologyand can be used to introduce any variation in any part of a given gene.

Ashraf M et al developed a randomization method of generating DNAcassettes for saturation mutagenesis, i.e., replacing of wild-typecodons with codons for all 20 amino acids, without degeneracy or bias(Biochem Soc Trans. (2013) 41(5):1189-94, which is incorporated hereinby reference). In short, double-stranded DNA donors, carrying randomizedcodon at their termini, are ligated individually on to a double-strandedDNA acceptor sequence, which is phosphorylated at the 5′ end only. Afterligation, the products are amplified, purified, quantified and thencombined in the required ratios. The combined product is digested withMlyI, which generates a double-stranded DNA consisting of the acceptorsequence plus the randomized codon at the 5′ end. The process is thenrepeated, using the double-stranded DNA product from the previous cycleas the acceptor for the next round of ligation. As a result, saturationmutagenesis can be introduced to contiguous codons.

The combination of the above methods and alike can generate any and allvariants of a target gene.

The variants of the target gene can be cloned to a nucleic acid vectorto generate a library of nucleic acid constructs that includes allvariants of a target gene. Suitable eukaryotic vectors from which onecan construct the nucleic acid constructs are well known in the art.See, for example, Broach, Cell (1982) 28:203-204; Dilon et al., J. Clin.Hematol. Oncol. (1980) 10:39-48; Maniatis, In: Cell Biology: AComprehensive Treatise, Vol. 3, Gene Sequence Expression, AcademicPress, NY, pp. 563-608, 1980.

The library of nucleic acid constructs is then introduced to the cellline to generate a cell library. The nucleic acid constructs can beintroduced to the cell line using methods known in the art, such astransformation or transfection. The recombinase that recognizes therecombination sites are expressed in the cell line, which mediates therecombination between the first and the second recombination sites,resulting in the incorporation of the variants of the target gene to thetarget genomic locus of the cell line. In some preferred embodiments,the concentration of the library of nucleic acid constructs is adjustedso that single variant is introduced into each cell. In someembodiments, the cell library can be enriched with a selection marker.

Selecting Desired Variant

A screening method can be designed to select desired variant of thetarget gene from the library of cells.

In one embodiment, where a target gene encodes an integrase, a screenmethod can be designed to select an integrase variant that recognizes avariant of recombination site (i.e., a mutant recombination site), e.g.,a pseudo-recombination site. In such a screen method, a selectionconstruct is generated to comprises: a third unidirectionalrecombination site, a third promoter, a fourth unidirectionalrecombination site, and a selectable marker (e.g., an antibioticresistance gene), wherein at least one of the third and the fourthunidirectional recombination sites is a variant or mutant that is notrecognized by the original integrase but recognized by the desiredvariant of the integrase, and wherein the third promoter and theselectable marker is arranged in opposite orientation.

The selection construct is then introduced to the cell library thatcomprises the variants of the integrase gene. The transformed celllibrary is maintained under conditions that facilitate recombinationbetween the third and the fourth unidirectional recombination sitesmediated by the desired variant of the integrase, thereby reversing theorientation of the third promoter or the selectable marker in theselection construct. The third promoter (or the selectable marker) afterreversing the orientation then direct the expression of the selectablemarker (or be directed by the third promoter when the selectable markerreverses the orientation). Therefore, when the transformed cell libraryis subject to the selectable condition, e.g., in the presence ofantibiotics, the cell containing the desired variant of the integrasecan be selected.

In another embodiment, where the target gene is a virus capsid gene(e.g., an AAV capsid gene), a screen method can be designed to select avariant that has larger packaging ability (e.g., packaging>4.7kb DNA insize) or has increased infectivity to a target cell/tissue. See, e.g.,Ojala D S et al. “In Vivo Selection of a Computationally Designed SCHEMAAAV Library Yields a Novel Variant for Infection of Adult Neural StemCells in the SVZ.” Mol Ther (2018) 26(1): 304-19, which is incorporatedby reference.

In another embodiment, where the target gene is a CRISPR/Cas protein. Ascreen method can be designed to select the variant of the Cas proteinthat recognizes a variant protospacer adjacent motif (PAM), or hashigher on-target specificity or has lower immunogenicity.

Stepwise Selection of Desired Variant

Theoretically, the method disclosed herein can generate any and alldesired variant by using a sufficiently large variant library. Inpractice, however, due to the size limit of the variant library, adesired variant may not be found in one round of selection if it isdifferent from the original gene/protein in too many positions, e.g.,nucleotide residues or amino acid residues. Therefore, in certainembodiments, the selecting method disclosed herein involves stepwiseselection of desired variant by generating a series of intermediatevariants, each intermediate variant is different in just a few positionsfrom the original gene or the intermediate variant gene generated in theprevious round of selection.

In one exemplary embodiment, the method disclosed herein selects avariant integrase (e.g., a phiC31 integrase) that can recognize a mutantattP sequence (e.g., SEQ ID NO: 3). The target mutant attP sequence isdifferent from the wildtype attP site in more than 50% of the nucleotideresidues, e.g., 26 out of 48 nucleotide residues are different. Toobtain a variant integrase that recognizes the target mutant attPsequences, an intermediate mutant attP sequences, e.g., a sequenceselected from SEQ ID NOS: 4-10, is created to identify an intermediatevariant integrase that recognizes the intermediate mutant attP sequencefrom a mutant integrase library. The identified intermediate variantintegrase gene is then used as the start integrase gene to generate amutant integrase library, which is used in the next round of selectionto identify variant integrase that recognizes the target mutant attPsequence or a second intermediate variant integrase that recognizes asecond intermediate mutant attP sequence more similar to the targetmutant attP as compared to the intermediate mutant attP sequence used inthe previous round of selection.

Kits and Compositions

The present disclosure in another aspect provides kits and compositionsfor screening a desired variant of a target gene. In one embodiment, thekit comprises: (i) a cell line comprising at a genomic locus: a firstunidirectional recombination site recognized by a site-specificunidirectional recombinase; (ii) a nucleic acid construct comprising asecond unidirectional recombination site recognized by the site-specificunidirectional recombinase; and (iii) a reagent for generating a variantlibrary of a target gene.

Components of such kits can include, but are not limited to, containers,instructions, solutions, buffers, disposables, and hardware. Reagentsfor generating a variant library of a target gene typically comprise oneor more nucleic acid constructs with instructional materials disclosingmeans of use these constructs in the procedures of building a variantlibrary to be employed in the present methods. The kits may also includeadditional components to facilitate the particular application for whichthe kit is designed (e.g., positive controls, negative controls, and/orreporter expression in vivo). The kits may additionally include buffersand other reagents routinely used for molecular biology, microinjection,and assessment by PCR, etc. Such kits and appropriate contents are wellknown to those of skill in the art.

The foregoing description of the invention has been presented forpurposes of illustration and description. It is not intended to beexhaustive or to limit the invention to the precise form disclosed.Other modifications and variations may be possible in light of the aboveteachings. The embodiments were chosen and described in order to bestexplain the principles of the invention and its practical application,and to thereby enable others skilled in the art to best utilize theinvention in various embodiments and various modifications as are suitedto the particular use contemplated. It is intended that the appendedclaims be construed to include other alternative embodiments of theinvention; including equivalent components, methods, and means.

It is appreciated that the Summary and Abstract sections may set forthone or more, but not all exemplary embodiments of the present inventionas contemplated by the inventor(s), and thus, are not intended to limitthe present invention and the appended claims in any way.

EXAMPLES Example 1

This example illustrates the high integration efficiency of a cell linecontaining a Bxb1 attP site at the H11 locus.

As shown in FIG. 1, an HEK293 cell line was created to contain a landingpad to receive heterologous genes at either intergenic Hipp1 (H11) locusor AAVS1 locus. The landing pad comprises a Bxb1 attP site flanked by aTet promoter and an EF-1 promoter.

A nucleic acid construct was transfected to the cell lines above. Thenucleic acid construct contained a Bxb1 attB site and a GFP gene. Uponthe expression of Bxb1 integrase in the cell lines, the recombinationbetween the Bxb1 attB and attP sites results in the incorporation of theGFP gene in the genome of the cell line under the control of the EF1promoter. The expression of the GFP gene was then assayed using flowcytometry.

As shown in FIG. 1 and Table 1 below, the cell line containing the Bxb1attP site at the H11 locus demonstrated higher integration efficiency ascompared to the AAVS1 locus. The increased integration efficiency ismore striking when compared to other systems reported, which had about8% integration efficiency. See Duportet X et al., “A platform for rapidprototyping of synthetic gene networks in mammalian cells.” NucleicAcids Res. (2014) 1; 42(21):13440-51; and Matreyek K et al., “A platformfor functional assessment of large variant libraries in mammalian cells”Nucleic Acids Res (2017) 45(11):e102.

TABLE 1 Bxb1 integration efficiency summary Locus Bxb1 integrase PercentGFP Median GFP Intensity AAVS1 − 0.74 3,092 AAVS1 + 15.4 26,802 H11 −0.85 2,888 H11 + 19.4 32,758

Example 2

This example illustrates the high integration efficiency using anoptimized Bxb1 integrase expression construct.

To increase the integration efficiency of a heterologous gene, a Bxb1expression construct was generated to contain a Bxb1 gene with optimizedcodon and a bi-partite Xenopus nuclear localization sequence (NLS) (seeSEQ ID NO: 1 in FIG. 2).

The Bxb1 expression construct was used to test the integrationefficiency using the HEK293 cell line (containing an attP site at theH11 locus) and the GFP report construct as described in Example 1.

As shown in FIG. 3, in the presence of VPA (valproic acid) atconcentration of 700 nM or 1 mM, the Bxb1 expression construct gave riseto an integration efficiency of 43.5% and 45.2%, respectively, muchhigher than the integration efficiency as previously reported (DuportetX et al., Nucleic Acids Res. (2014) 1; 42(21):13440-51; and Matreyek Ket al., Nucleic Acids Res (2017) 45(11):e102). In the absence of VPA,the Bxb1 expression construct gave rise to an integration efficiency of27.6% (compared to the integration efficiency of 42.7% with 1 mM VPA ina side-by-side experiment).

Example 3

This example illustrates a method of screening a variant of phiC31integrase that recognizes a mutant attP site.

The target mutant attP site was selected based on the followingcriteria: 1) it has a GC content of 20-60%; 2) it is symmetricallysimilar to the wildtype phiC31 attP site; 3) it is unique in the humangenome; 4) it is intergenic; 5) it is not near to oncogene oranti-oncogene; and 6) it is close to high expression region or DNase Ihypersensitive site. Under these criteria, Site A of SEQ ID NO: 3 (FIG.4) was selected.

Compared to the wildtype attP sequence, which has 48 nucleotide residuesto be recognized by the wild type phiC31 integrase, Site A is identicalin 22 nucleotide residues. Considering that it may be difficult todirectly screen a phiC31 integrase variant that recognizes Site A in oneround of screening, a series of intermediate Site A sequences (SEQ IDNOS: 4-10) was generated for screening intermediate phiC31 integrasevariants.

A library of mutant phiC31 integrase gene for selecting a mutant phiC31integrase that recognizes intermediate Site A 4, A12 and A13 was thengenerated using repeated error prone PCR, two-codon saturation withProxiMAX and hybridization. As shown in FIG. 5, each of the nucleic acidconstruct in the mutant phiC31 library contains a Bxb1 attB site, ablasticidin resistance marker and a mutant phiC31 integrase gene.

The library was then transformed to the cell line as described inExample 1 to generate a cell library. The Bxb1 expression construct asdescribed in Example 2 was introduced to the cell library to express theBxb1 integrase. As shown in FIG. 6, upon the expression of Bxb1integrase, the recombination between the Bxb1 attB and attP sitesresulted in the incorporation of a mutant phiC31 integrase in the H11locus under the control of the Tet promoter and the blasticidinresistance gene under the control of the EF-1 promoter.

To screen a mutant phiC31 integrase that recognizes a particularpseudo-phiC31 attP site, as shown in FIG. 7, a selection construct thatcontains a CMV promoter and a GFP reporter gene was generated. The CMVpromoter is flanked by the pseudo-phiC31 attP site and a phiC31 attBsite. The CMV promoter and the GFP reporter gene is in oppositedirection so that the GFP reporter gene is not expressed when there isno recombination between the pseudo-phiC31 attP site and the phiC31 attBsite.

The selection construct was then transfected to the cell library. Asshown in FIG. 7, when a cell containing the variant phiC31 integrasethat recognizes the pseudo-attP site, a recombination between thepseudo-phiC31 attP site and the phiC31 attB site occurs and theorientation of the CMV promoter in the selection construct is reversed.The reversed CMV promoter then drives the expression of the GFP reportergene. The GFP positive cells were then isolated via FACS or chitin-beadbinding. The integrases present in the isolated cells were amplified andanalyzed. Potential phiC31 integrase variants that recognizeintermediate Site A12 were enriched and identified, which was used toperform the next round of selection to identify variants that recognizethe Site A.

Example 4

This example illustrates the screening of a mutant AAV capsid thatbetter packages therapeutic cassette of large DNA size (e.g., BDDFVIII).

As shown in FIG. 8, a HEK293 cell line that contains at H11 locus alanding pad as described in Example 3 is generated.

A nucleic acid construct library (donors) is then transformed to thecell line to generate a cell library. Each of the nucleic acid constructcontains a Bxb1 attB site, a blasticidin resistance marker, a mutantcapsid gene, and a therapeutic cassette (e.g., BDD FVIII cassette). Uponthe expression of Bxb1 integrase in the cell line, the recombinationbetween the Bxb1 attB and attP sites results in the incorporation of amutation capsid gene in the H11 locus under the control of the Tetpromoter together with the therapeutic cassette.

To screen the desired mutant capsid gene, the cell line is transfectedwith helper construct to generate viruses that packaging the therapeuticcassette. The viruses generated are infected to target cells/tissues,and those having higher infection efficiency are amplified and enriched.The cells/tissues are collected to extract DNAs that contain the desiredmutant capsid gene.

What is claimed is:
 1. A method of screening for a desired variant of atarget site-specific recombinase, the method comprising: (1) obtaining afirst plurality of mammalian cells each of which comprises at a genomiclocus (i) a first recombination site recognized by a serine integrase,wherein the serine integrase is different from the target site-specificrecombinase and (ii) a first promoter and a second promoter; (2)generating a cell library by (i) introducing into the first plurality ofmammalian cells a library of nucleic acid constructs, each of thenucleic acid constructs comprising: (a) a second recombination siterecognized by the serine integrase (b) a polynucleotide encoding avariant of the target site-specific recombinase, and (c) a firstselectable marker, thereby generating a second plurality of mammaliancells comprising the library of nucleic acid constructs, wherein atleast one of the nucleic acid constructs comprises a polynucleotideencoding the desired variant of the target site-specific recombinase,(ii) expressing in each of the second plurality of mammalian cells theserine integrase, and (iii) maintaining the second plurality ofmammalian cells expressing the serine integrase under conditions thatfacilitate recombination between the first and the second recombinationsites mediated by the serine integrase, thereby generating a celllibrary comprising a third plurality of mammalian cells each of whichcomprises a variant of the target site-specific recombinase at thegenomic locus, wherein the first promoter drives the expression of thevariant of the target site-specific recombinase and the second promoterdrives the expression of the first selectable marker; (3) selecting,from the cell library, a mammalian cell expressing the desired variantof the target site-specific recombinase by (i) introducing a selectionconstruct to each mammalian cell of the cell library, wherein theselection construct comprises (a) a third recombination site, (b) afourth recombination site, and (c) a second selectable marker, whereinat least one of the third and the fourth recombination sites are notrecombined by the target site-specific recombinase but are recombined bythe desired variant of the target site-specific recombinase, and whereinthe second selectable marker is not expressed in the absence of arecombination between the third and the fourth recombination sites inthe mammalian cell, and (ii) maintaining the cell library with theselection construct under conditions that facilitate recombinationbetween the third and the fourth recombination sites mediated by thedesired variant of the target site-specific recombinase, and (iii)detecting expression or the lack of expression of the second selectablemarker that indicates the recombination between the third and the fourthrecombination sites in the mammalian cell, and (4) generating from themammalian cell selected in step (3) a polynucleotide encoding thedesired variant of the target site-specific recombinase.
 2. The methodof claim 1, wherein the genomic locus is Hipp11 (H11) locus.
 3. Themethod of claim 1, wherein the serine integrase is Bxb1 integrase,phiC31 integrase, TP901-1 integrase, R4 integrase, gamma-deltaresolvase, Tn3 resolvase, or Gin invertase.
 4. The method of claim 3,wherein the serine integrase is the Bxb1 integrase encoded by thepolynucleotide sequence of SEQ ID NO:
 1. 5. The method of claim 1,wherein the first promoter is a Tet-on promoter.
 6. The method of claim1, wherein the second promoter is an EF-1 alpha promoter.
 7. The methodof claim 1, wherein the first plurality of mammalian cells are HEK293cells.
 8. The method of claim 1, wherein the target site-specificrecombinase is phiC31 integrase, Bxb1 integrase, TP901-1 integrase, R4integrase, gamma-delta resolvase, Tn3 resolvase, Gin invertase, Crerecombinase or Flp recombinase.
 9. The method of claim 1, wherein theselection construct further comprises (d) a third promoter, wherein thethird promoter is arranged in an opposite orientation of the secondselectable marker, and wherein the recombination between the third andthe fourth recombination sites reverse the orientation of the thirdpromoter or the second selectable marker in the selection construct,thereby the third promoter driving the expression of the secondselectable marker.
 10. The method of claim 1, wherein the firstselectable marker is neomycin resistance gene, a puromycin resistancegene, a hygromycin resistance gene, or a blasticidin S resistance gene.11. The method of claim 1, wherein the second selectable marker is a GFPor RFP gene.